Studies of Direct Pluripotent Stem Cell Programming. It is widely known that the induction of cell fates during embryogenesis is orchestrated through the action of developmental signals, such as growth factors (1). Proper exposure to these signals leads to the normal patterning and growth of the embryo. We recently proposed that one outcome of this signaling, cell type differentiation, involves among other factors alignment of the transcriptional or epigenetic state, which determines the cell type to be specified, and 2. It's appropriate cell cycle state, which affects the rate of differentiation (2). Within some limits, t may be possible to perturb the normal process of differentiation and achieve differentiated states more rapidly and through pathways that do not occur naturally. This study of the alteration of the normal differentiation process helps us understand the elements required for cell fate and cannot be understood by studying only normal embryonic differentiation. Furthermore, these altered pathways of differentiation could lead to useful approaches for regenerative medicine. The aim of this proposal is to develop and to better understand the pathway independence of differentiation and the special role of transcriptional regulators and cell cycle regulation. We will address three important issues: 1. What are the underlying molecular changes that underlie altered pathways of differentiation, 2. In terms of different cell types, how widely applicable are the methods we developed, and 3. Can these discoveries in mouse ES cells be demonstrated in human.